Genetic materials, procedure, and SSR markers

A total of 63 yellow-seeded soybean genotypes collected from the BB-Biogen National Gene Bank (Table 1) were used in this study. The genetic materials were planted at the Cikeumeuh Experimental Station, Bogor, West Java, from March to June 2023. The experiment was conducted under field conditions to evaluate the morphological characteristics, including the yield component traits, of the genotypes.

Field evaluation was conducted using a randomized complete block design with three replications. The genotypes were grown following standard soybean cultivation practices (^{Asadi et al., 2020}). Each genotype was planted in a 2 m × 3 m plot with a spacing of 40 cm × 15 cm, with two seeds per hill. Prior to planting, the field was thoroughly prepared, and organic manure was applied at a rate of 1 t ha^-¹ to improve soil fertility. Inorganic fertilizers consisting of 50 kg ha^-¹ urea, 150 kg ha^-¹ SP36, and 100 kg ha^-¹ KCl were applied at planting. Irrigation, weed control, pest management, and disease control were carried out according to recommended soybean cultivation practices to ensure optimal plant growth.

Molecular marker analysis was conducted at the Genomic Laboratory, BRIN, Cibinong, West Java, from January to March 2024. The SSR markers used were selected based on their broad distribution across the soybean genome and their proven effectiveness in genetic diversity studies (Table 2). These markers were selected to provide representative genome-wide coverage across soybean chromosomes (^{Tasma et al., 2018}).

Genomic DNA isolation, qualitative and quantitative analysis

Genomic DNA was isolated from young leaves following the method described by ^{Doyle and Doyle (1990}) with a minor modification consisting of the addition of 2 % (w/v) polyvinylpyrrolidone (PVP). The dried DNA pellets were subsequently dissolved in 100 µL 1× TE buffer (10 mM Tris (pH 8.0), 1 mM EDTA), supplemented with 2 µL of 10 mg/mL RNase, and incubated at 37 °C for one hour. DNA quality was assessed by running genomic DNA on a 1 % agarose gel, whereas genomic DNA concentration was determined using a NanoDrop 2000 spectrophotometer.

PCR and electrophoresis

PCR amplification of the 63 soybean genotypes was performed using 14 SSR markers (Table 2). Each PCR reaction was carried out in a total volume of 10 µL, consisting of 40 ng template DNA, 5 µL PCR master mix, 0.5 µL each of 10 µM forward and reverse primers, and 2 µL sterile ddH2O to a final volume of 10 µL.

The PCR program consisted of an initial DNA denaturation step at 94 °C for 30 s, followed by primer annealing at 55 °C for 1 min, DNA extension at 72 °C for 1 min, and final extension at 60 °C for 15 min, and the reaction was completed by incubating the PCR products at 10 °C for 4 min.

PCR products were separated by electrophoresis on 8 % polyacrylamide gels for 120 min at 90 V using 1 × TBE (Tris Borate EDTA) buffer. DNA fragments were stained with ethidium bromide and visualized using a Gel Doc Transilluminator system. The resulting DNA banding patterns were subsequently scored by using appropriate gel analysis software.

Morphological and yield component data analysis

The morphological traits analyzed included hilum color, flowering time, maturity time, plant height, number of branches per plant, number of pods per plant, 100-seed weight, and seed yield per plant. The qualitative trait hilum color was converted to numerical data according to the Union for the Protection of New Varieties of Plants (UPOV, 1998) classification system as follows: 1 = gray, 2 = yellow, 3 = light brown, 4 = dark brown, 5 = dull black, 6 = black, and 7 = brown. All morphological and yield component data were subjected to principal component analysis (PCA) and principal coordinate analysis (PCoA) using R software, version 3.6.3.

Molecular data analysis

Molecular data analysis was based on the scoring of DNA banding patterns obtained from electrophoresis of the SSR marker PCR products on 8 % polyacrylamide gels. Band scoring was performed using GelAnalyzer Software v.2010a (^{Lazer & Horvath-Lazar, 2010}). DNA fragments exhibiting identical migration patterns and the same amplicon sizes (bp) were considered to represent homologous loci. The presence of a DNA band was scored as 1, whereas its absence was scored as 0, generating a binary data matrix for subsequent analysis.

The scored data were analyzed using the sequential agglomerative hierarchical and nested (SAHN) clustering procedure of the unweighted pair group method with arithmetic mean (UPGMA) implemented in NTSYS-pc software version 2.1 (^{Rohlf, 2000}) to generate a dendrogram of the soybean genotypes. PowerMarker V3.25 (^{Liu & Muse, 2005}) was also used to analyze the marker statistics, such as polymorphism information content (PIC) values, main allele frequency, gene diversity, and heterozygosity level of the SSR markers.

Morphological characteristic analysis

A total of eight morphological traits were evaluated in 63 yellow-seeded soybean genotypes included in this study. The traits assessed were hilum color, flowering time, maturity time, plant height, number of branches per plant, number of pods per plant, 100-seed weight, and seed yield per plant. The descriptive statistics for these traits are presented in Table 3.

The hilum color observed among the soybean genotypes was classified into four categories: light brown (23 genotypes), dark brown (32 genotypes), brown (3 genotypes), and black (5 genotypes). Flowering time ranged from 36 to 43 days after planting (DAP) with a mean value of 39 DAP. Maturity time varied from 86 to 89 DAP, averaging 87 DAP. Plant height ranged from 64 to 154 cm with a mean of 97 cm. The number of branches per plant varied from 2 to 6 with an average of 4 branches per plant. The number of pods per plant ranged from 56 to 137 with a mean of 85 pods per plant. The 100-seed weight varied from 6.00 to 19.40 g, averaging 10.69 g. Seed yield per plant ranged from 9.38 to 30.48 g with an average of 16.86 g per plant.

PCA reduced the morphological traits in the 63 soybean genotypes into four principal components with eigenvalues greater than 1 which together explained 72.63 % of the total phenotypic variation (Table 4). The first principal component (PC1) was primarily associated with flowering time, number of branches per plant, and number of pods per plant, along with other contributing traits, and accounted for 26.26 % of the total phenotypic variation. The second principal component (PC2) was mainly associated with 100-seed weight and seed yield per plant, along with other contributing traits, explaining 19.72 % of the total phenotypic variation. The third principal component (PC3) was predominantly influenced by plant height and associated traits, accounting for 13.99 % of the total phenotypic variation. The fourth principal component (PC4) was primarily associated with maturity time and related traits, explaining 12.77 % of the total phenotypic variation.

Figure 1A presents the PCA biplot of morphological traits evaluated in the 63 soybean genotypes. The first principal component (PC1) and the second principal component (PC2) accounted for 26.2 % and 19.7 % of the total phenotypic variation, respectively, consistent with the values shown in Table 4. Based on Figure 1A, PC1 was characterized by the number of branches per plant (0.72), number of pods per plant (0.71), and flowering time (0.54), indicating that these traits contributed most strongly to the variation explained by this component. This pattern is clearly reflected in the biplot, where these variables are represented by relatively long vectors oriented in similar directions.

Figure 1 Principal component analysis of morphological traits in 63 yellow-seeded soybean (Glycine max) genotypes evaluated at Cikeumeuh Experimental Station, Bogor, Indonesia, from March to June 2023. A) Distribution pattern of morphological trait diversity based on the first and second principal components. B) Relative position of soybean genotypes based on morphological traits. 

The length of the vectors reflects the magnitude of trait contribution to the principal components, with longer vectors indicating stronger contributions, while shorter vectors indicate weaker contributions. Furthermore, the small angles (<90°) formed among these vectors suggest strong positive correlations among the traits. Similarly, PC2 was characterized by seed yield per plant (0.74) and 100-seed weight (0.57), as shown in Table 4. In the biplot, these traits were also represented by long vectors with similar orientations, indicating their substantial contribution to PC2 and their positive association.

The PCoA illustrating the relative position of the 63 soybean genotypes based on morphological traits can be used to select plant genotypes based on a two-dimensional space (Figure 1B). The genotypes were distributed across the four quadrants, with several groups of genotypes clustering closely together. Genotypes located in close proximity exhibit greater similarity in morphological characteristics.

For example, genotype 21 (Lokal Karangasem) and genotype 57 (Lokal Klungkung) were positioned within the same quadrant and clustered closely together (Figure 1B). The two genotypes originated from the same island, Bali (Table 1), and exhibited similar morphological characteristics, particularly hilum color, flowering time, and maturity time (Table 3).

Soybean genotypes located in close proximity within the same quadrant are less suitable as parental candidates because they are more likely to share similar morphological traits and possess a similar genetic background, which may result in inferior progenies. Based on this study, genotype 8 (Kepet) and genotype 44 (Sinabung) appear to be promising parental candidates. These genotypes were positioned far apart in the ordination plot and were located in different quadrants (Figure 1B). Furthermore, the two genotypes differed in multiple morphological traits, including hilum color (Table 3). Therefore, these genotypes can be considered morphologically divergent and potentially valuable parental materials for soybean breeding programs because their genetic divergence may contribute to the development of superior progenies.

Molecular marker analysis

Molecular marker analysis using 14 SSR markers revealed high levels of polymorphism among the 63 soybean genotypes, with amplified fragment sizes ranging from 89-240 bp (Figure 2; Table 5). The polymorphism information content (PIC) values ranged from 0.88 (Satt063) to 0.98 (Satt191) with a mean of 0.95. A total of 553 SSR alleles were detected across all SSR loci with an average of 40 alleles per locus and a range of 26 to 57 alleles per locus.

Figure 2 Representative SSR banding profile generated by marker Satt191 in 63 yellow-seeded soybean (Glycine max) genotypes, at the Genomic Laboratory, BRIN, Cibinong, Indonesia, from January to March 2024. 

The major allele frequencies ranged from 0.06 for markers Satt002, Satt045, Satt191, Satt197, Satt294, and Satt308 to 0.25 for marker Satt063, with an average value of 0.10. Gene diversity values ranged from 0.89 (Satt063) to 0.98 (Satt191), with a mean of 0.95. Observed heterozygosity ranged from 0 (Satt030) to 1.00 (Satt045 and Satt191), with an average value of 0.65 (Table 5).

Phylogenetic analysis

The analysis of genetic relationships among the 63 soybean genotypes based on 14 SSR markers revealed an overall genetic similarity of 76 % (Figure 3). Using this similarity threshold, the soybean genotypes were classified into two major clusters, namely Cluster I, comprising 39 genotypes, and Cluster II, comprising 24 genotypes.

Figure 3 Dendrogram of 63 yellow-seeded soybean (Glycine max) genotypes based on the results of analysis using 14 SSR markers, generated at the Genomic Laboratory, BRIN, Cibinong, Indonesia, from January to March 2024. 

As shown in Figure 3, soybean genotypes originating from the same geographic region and exhibiting similar hilum color tended to cluster together. For example, three accessions originating from Bali, namely Balinese soybean, Lokal Klungkung, and Lokal Tabanan, were grouped together in Cluster I. Similarly, four local accessions from Subang (Kepet, Kedelai Godek, Kedelai Minyak, and Kedelai Susu) were grouped in Cluster II.

In several cases, clustering patterns were also associated with hilum color. For example, genotypes C.7301-1113-4-C-pop and GM 1461 Si, both characterized by brown hilum color, were grouped together in Cluster I-b. Likewise, four accessions with dark brown hilum color and a flowering time of 40 days (Lokal Jember, Lokal Karangasem, MLG 2830, and Lokal West Lombok) were grouped together in Cluster II-a (Figure 3).